Methods and apparatus for conditioning cell populations for cell therapies

ABSTRACT

A bioreactor system for conditioning of pluripotent cells or cell media is provided. In further aspects, conditioned pluripotent cells and methods for making such cells are provided.

This application is a divisional of U.S. patent application Ser. No.15/738,257, filed Dec. 20, 2017, as a national phase application under35 U.S.C. § 371 of International Application No. PCT/US2016/039044,filed Jun. 23, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/183,273, filed Jun. 23, 2015, the entirety of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

Embodiments of the present invention relate to methods and apparatus forconditioning cell populations for improved characteristics for use astherapeutic agents. More specifically, the embodiments of the presentinvention relate to an apparatus and a method for conditioning stemcells by imposing a controlled shear stress on stem cells disposed alonga boundary of a flow chamber in which the stem cells are disposed.

Background of the Related Art

Lineage specific differentiated cell populations are contemplated foruse in cell replacement therapies for patients with diseases ordisorders. Cell populations that retain the ability to differentiateinto specialized cell types (stem cells) and/or secrete certain factorshave been contemplated for use in cell-based therapies for patients witha variety of diseases or disorders.

Research and technological developments relating to directeddifferentiation of stem cells has been postulated to provide treatmentsfor many diseases and disorders. However, there is still a need for theability to obtain sufficient donor cell populations that are reliablyconditioned such that they are predictable in their therapeuticactivity. The present methods and apparatus provide a solution to theseproblems and thus facilitate the use of cells as cellular therapeuticsor as the source of soluble factors.

A bioreactor is a device in which components of biological materials,such as stem cell-containing fluids, may be conditioned by manipulationof the factors that influence the materials. The condition of a stemcell-containing fluid is influenced by multiple factors including pH,waste content, nutrient content and the type and concentration ofdissolved gases such as, for example, oxygen. These factors maygenerally be referred to as chemical factors that influence thecondition of stem cells in a stem cell-containing fluid.

A bioreactor may enable the manipulation of the condition of the stemcell-containing fluids by control of non-chemical factors. Conventionalapparatuses and methods of conditioning cell populations forconventional cell therapies fail to enable the precise control ofmechanical shear to the cells to be conditioned. Apparatus and methodsfor controllably applying shear stress to cells to be conditioned withina bioreactor flow chamber capable of large-scale cell production aretherefore desired.

SUMMARY OF THE INVENTION

Embodiments of the present invention include apparatus that enable thecontrol of at least one mechanical factor that influences the conditionof the stem-cell containing fluids. More specifically, embodiments ofthe present invention include apparatus that enable the control of therate of shear stress applied to stem cells within the stemcell-containing fluid.

Embodiments of the present invention may be used to flow a media fluidthrough one or more flow chambers shaped to provide a very high flowboundary layer perimeter to cross-sectional flow area ratio to therebymaximize the conditioning effects of mechanical forces (shear) on stemcells adherent to the chambers.

The very high boundary layer perimeter for a correspondingcross-sectional flow area is calculated, for a given flow passage, asthe perimeter of the flow chamber divided by the cross-sectional flowarea. For example, a circular flow passage provides the minimumachievable flow boundary layer perimeter for a correspondingcross-sectional flow area, which is calculated as: 2πr÷πr²=2/r. It willfollow that a flow passage having a minimal perimeter to cross-sectionalflow area ratio generally provides for minimal shear stress imparted tothe fluid as it flows through the flow passage, all other factors(turbulent or laminar flow regime, surface roughness, etc.) being thesame.

The maximum flow boundary layer perimeter for a correspondingcross-sectional flow area is provided by, for example, an infinitelywide and infinitesimally thin passage, for which the achievable flowboundary layer perimeter for a corresponding cross-sectional flow areatheoretically approaches Go. Of course, an infinitely wide andinfinitesimally narrow passage is impractical as it would imposeinfinite resistance to flow, but a very high flow boundary layerperimeter for a corresponding cross-sectional flow area is achieved byproviding wide, but thin flow passages to promote the application ofshear along the flow boundary layer perimeter to a stem cell-containingfluid moved therethrough.

One embodiment of an apparatus of the present invention provides anapparatus, comprising a first cap having a first end, a second end, awall therebetween, an inlet fluid connector proximal to the first end, ariser bore proximal to the first end, and feed passage extending fromthe inlet fluid connector to the riser bore to dispose the riser bore influid communication with the inlet fluid connector, a second cap havinga first end, a second end, a wall therebetween, an outlet fluidconnector proximal to the second end, a riser bore proximal to thesecond end, and a drain passage extending from riser bore to the outletfluid connector to dispose the riser bore in fluid communication withthe outlet fluid connector, and at least one intermediate moduledisposed intermediate the wall of the first cap and the wall of thesecond cap and in sealed engagement with each of the first cap and thesecond cap upon assembly of the apparatus, each intermediate moduleincluding a frame having a first end, a second end, a barriertherebetween having a first side disposed toward the wall of the firstcap and a second side disposed toward the wall of the second cap, thebarrier disposed within the frame and between the first end and thesecond end to form a first flow chamber between the wall of the firstcap and the barrier of the intermediate module upon sealable engagementof a sealing face of the first cap with a sealing face of a first sideof the intermediate module and to also form a second flow chamberbetween the wall of the second cap and the barrier of the intermediatemodule upon sealable engagement of a sealing face of the second cap witha sealing face of a second side of the intermediate module with asealing face of the second cap with the intermediate module, a modulefeed bore in the frame proximal to the first end of the intermediatemodule for sealably engaging the riser bore of the first cap uponassembly of the apparatus, a distribution channel in the frame proximalto the first end of the intermediate module and in fluid communicationwith the module feed bore for distributing a fluid flow received intothe distribution channel from the module feed bore, a plurality of flowchamber feed ports disposed proximal to the first end of theintermediate module and in fluid communication with the distributionchannel for introducing a fluid flow received into the first and secondflow chambers, a plurality of flow chamber drain ports disposed proximalto the second end of the intermediate module and in fluid communicationwith the first and second flow chambers, a gathering channel disposedalong the second end of the intermediate module and in fluidcommunication with the first and second flow chambers through the flowchamber drain ports, a module drain bore proximal to the second end ofthe intermediate module and in fluid communication with the gatheringchannel and for sealably engaging the riser bore of the second cap uponassembly of the apparatus, wherein upon assembly of the apparatus withthe one or more intermediate modules disposed intermediate the first capand the second cap to sealably engage the first side sealing surface onthe one or more intermediate modules with the sealing surface on thefirst cap and to also sealably engage the second side sealing surface onthe one or more intermediate modules with the sealing surface on thesecond cap disposes the inlet fluid connector of the first cap in sealedengagement with the outlet fluid connector on the second cap to providea bifurcated fluid path that includes the first flow chamber, disposedintermediate the at least some of the plurality of flow chamber feedports and at least some of the plurality of flow chamber drain ports andintermediate the at least one intermediate module first side and thefirst cap, and a second flow chamber, disposed intermediate the at leastsome of the plurality of flow chamber feed ports and at least some ofthe plurality of flow chamber drain ports and intermediate the at leastone intermediate module second side and the second cap, wherein theplurality of flow chamber feed ports distributed along the distributionchannel and the plurality of flow chamber drain ports distributed alongthe gathering channel of the intermediate module provide for a moreuniform localized fluid velocity within the flow chambers upon providinga fluid pressure differential across the inlet fluid connector and theoutlet fluid connector of the apparatus, wherein a ratio of theperimeter of the first flow chamber to the cross-sectional flow area ofthe first flow chamber exceeds.

Certain embodiments of the invention concern a method of producing aconditioned composition. As used herein, a conditioned compositionrefers to a composition that has been subjected to the conditioningeffects of mechanical forces. For example, the mechanical force can bean application of controlled shear stress with a force sufficient toproduce a conditioned composition. In some aspects, the conditionedcomposition comprises a population of conditioned pluripotent cells(e.g., MSCs). Thus, certain aspects concern the isolation of apopulation of conditioned pluripotent cells. In further aspects, theconditioned composition is a media (e.g. a cell-free media) comprisingsecreted factors from pluripotent cells that have been subjected to acontrolled sheer stress.

Aspects of the embodiments involve culturing of the stem cells on asubstrate to allow cell adhesion. In some cases, the substrate is asurface that supports the growth of the stem cells in a monolayer. Forexample, in some aspects, the surface is a plastic or glass surface,such as a surface that has been coated with extracellular matrixmaterials (e.g., collagen IV, fibronectin, laminin and/or vitronectin).In further aspects, the substrate may be modified to incorporate asurface (or surface coating) with increased or decreased surface energy.Examples of low energy materials that may be used as a surface orsurface coating include, without limitation, hydrocarbon polymers, suchas polyethylene, or polypropylene and nitrides. For instance, a surfaceor surface coat may comprise Polyhexafluoropropylene,Polytetrafluoroetylene, Poly(vinylidene fluoride),Poly(chlorotrifluoroethylene), Polyethylene, Polypropylene,Poly(methylmethacrylate)—PMMA, Polystyrene, Polyamide, Nylon-6,6,Poly(vinylchloride), Poly(vinylidene chloride), Poly(ethyleneterephthalate), Epoxy (e.g., rubber toughened or amine-cured),Phenol-resorcinol resin, Urea-formaldehyde resin, Styrene-butadienerubber, Acrylonitrile-butadiene rubber and/or Carbon fibre reinforcedplastic. Examples of high energy materials that may be used as a surfaceor surface coating include, without limitation, metals and oxides. Forinstance, a surface or surface coat may comprise Aluminium oxide,Berylium oxide, Copper, Graphite, Iron oxide (Fe 2O 3), Lead, Mercury,Mica, Nickel, Platinum, Silicon dioxide—silica and/or Silver.

Further aspects of the embodiments concern the application of controlledshear stress with a force sufficient to produce a conditionedcomposition. In certain aspects, the shear stress is applied in the formof fluid laminar shear stress. For example, the force of the shearstress is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 dynes persquare centimeter. In certain instances, the force of the shear stressis at least 5, 10 or 15 dynes per square centimeter, such as betweenabout 5-20; 5-15; 10-20 or 10-15 dynes per square centimeter. In someaspects, the controlled shear stress is applies for a period of betweenabout 1 minute and two days. For example, the controlled shear stresscan be applied for a period of between 5 minutes to 24 hours; 10 minutesto 24 hours; 0.5 hours to 24 hours or 1 hour to 8 hours. In furtheraspects, cells of the embodiments are exposed to an elevated pressure.

In still further aspects, cells or media from a culture of theembodiments are tested periodically to determine the level ofconditioning. For example, a sample comprising cells or media can betaken from the culture about every 10 minutes, 15 minutes, 30 minutes orevery hour. Such samples may be tested, for example to determine theexpression level of anti-inflammatory factors, such as transcriptionfactors or cytokines. In particular aspects, cells or media can be takenfrom a sampling port positioned in a location of lower fluid pressure,including for example near the inlet of a pump configured to directfluid through the apparatus.

In certain aspects, a starting population of stem cells is obtained. Forexample, the starting stem cell population can comprise inducedpluripotent stem (iPS) cells or mesenchymal stem cells (MSCs). In someaspects, the MSCs are isolated from tissue. For example, in someaspects, the tissue comprises bone marrow, cord blood, peripheral blood,fallopian tube, fetal liver, lung, dental pulp, placenta, adiposetissue, or amniotic fluid. In further aspects the cells are human cells.For example, the cells can be autologous stem cells. In some aspects,the stem cells are transgenic cells.

In further aspects of the embodiments, a method of producing aconditioned composition comprises passing fluid over the stem cells forthe application of controlled shear stress. For example, the fluidpassed over the stem cells can be a cellular growth medium. In someaspects, the growth medium comprises at least a first exogenouscytokine, growth factor, TLR agonist or stimulator of inflammation. Forexample, the growth medium can comprise IL1B, TNF-α, IFNγ, PolyI:C,lipopolysaccharide (LPS), phorbol myristate acetate (PMA) and/or aprostaglandin. In certain specific aspects, the prostaglandin is16,16′-dimethyl prostaglandin E2 (dmPGE2).

In certain aspects, conditioned stem cells of the embodiments have atleast 2-, 3-. 4-. 5- or 6-fold higher expression of an anti-inflammatorygene compared to the starting populating of stem cells. For example, theanti-inflammatory gene can be TSG-6, PGE-2, COX-2, IL1Ra, HMOX-1, LIF,and/or KLF2.

In further embodiments, the conditioned composition comprises aconditioned media composition. Thus, certain aspects concern theisolation of conditioned media after the application of shear stress. Insome cases, the conditioned media is essentially free of cells.

Further aspects of the embodiments concern the application of shearstress in a bioreactor (e.g., such as a bioreactor detailed herein). Incertain aspects, the bioreactor system comprises an inlet fluid feedplate, a base plate, and a plurality of intermediate plates positionedbetween the inlet fluid feed plate and the base plate. For example, theintermediate plate comprises a first end, a second end, a first side,and a second side; a distribution channel proximal to the first end; anda gathering channel proximal to the second end. In further aspects, thedistribution channel extends between the first side and the second sideof the intermediate plate. In certain aspects, the gathering channelextends between the first side and the second side of the intermediateplate. In some cases, the bioreactor system further comprises areservoir, a first conduit, and a second conduit. In certain aspects,the inlet fluid feed plate comprises a first fluid inlet, a first fluidoutlet, a second fluid inlet and a second fluid outlet. For example, thereservoir is coupled to the first fluid inlet via the first conduit andis coupled to the second fluid outlet via the second conduit.

In further aspects of the embodiments, the bioreactor system comprises apump coupled to the first fluid outlet and the second fluid inlet of theinlet fluid feed plate. In certain aspects, the pump is configured todraw a fluid from the reservoir through first fluid inlet and the firstfluid outlet and to direct the fluid through the second fluid inlet,across a plurality of intermediated plates, out the second inlet, andback to the reservoir. In some aspects, a velocity of the fluid throughthe second fluid inlet is controlled by a rotational speed of therolling element of the pump. In certain aspects, the bioreactor systemprovides a high-flow boundary layer perimeter to cross-sectional flowarea ratio. In some aspects, the bioreactor system is capable oflarge-scale cell production. For example, the method of producing aconditioned composition is automated.

Certain aspects of the embodiments provide a composition of atherapeutically effective amount of conditioned stem cells. Some aspectsprovide a method of treating a subject in need of such treatmentcomprising administering a therapeutically effective amount ofconditioned stem cells. For example, the cells can comprise aconditioned composition (e.g., a cell composition or conditioned media)obtained by a method in accordance with the embodiments. As demonstratedherein such conditioned compositions can, in some aspects, provide forenhanced engraftment of stem cells in a subject. For example, in someaspects, the subject has chronic or acute inflammation,graft-versus-host disease, a neurological injury (e.g., an ischemicinjury, such as stroke, or a traumatic brain injury), musculoskeletaltrauma, or an autoimmune disorder. In some aspects, the conditioned stemcells are administered in combination with one or more additionaltherapeutic agents.

In still a further embodiment there is provided a method of increasingCD105⁺ cells in the bone marrow of a patent comprising administering aneffective amount of stem cells (e.g., mesenchymal stem cells) to thepatient. For example, the cells can comprise a conditioned composition(e.g., a cell composition or conditioned media) obtained by a method inaccordance with the embodiments. In some aspects, the patient has aneurological injury, such as an ischemic injury (e.g., from a stroke) ora traumatic brain injury. In certain aspects, the patient has sufferedfrom the neurological injury less than 24, 18, 12 or 6 hours before theadministration of the cells.

Certain aspects, the embodiments concern administration of cells and/orconditioned compositions. In some aspects, the administration can belocal, such as in or adjacent to, diseased or damaged tissues. Infurther aspects, the administration is systemic. For example, theadministration can be by injection of a composition, such as intravenousinjection.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and advantages of the present methods, system and apparatuswill be better understood and more readily apparent when considered inconjunction with the following detailed description and accompanyingdrawings which illustrate, by way of example, preferred embodiments ofthis system and methods.

FIG. 1 is a plan view of a feed cap of the bioreactor case according tothe present disclosure.

FIG. 2 is a side elevation view of the feed cap of FIG. 1.

FIG. 3 is a sectional elevation view of the feed cap of FIGS. 1 and 2taken through the riser bore.

FIG. 4 is an end view of the feed cap of FIGS. 1-3.

FIG. 5 is a plan view of an intermediate module of the bioreactor caseaccording to the present disclosure.

FIG. 6 is a side elevation view of the intermediate module of FIG. 5.

FIG. 7 is an enlarged view of the fluid passages at a second end of theside elevation view of the intermediate module of FIG. 6.

FIG. 8 is an end view of the second end of the intermediate module ofFIGS. 5-7.

FIG. 9 is a plan view of a drain cap of the bioreactor case of thepresent invention.

FIG. 10 is a side elevation view of the drain cap of FIG. 10.

FIG. 11 is a sectional elevation view of the drain cap of FIGS. 10 and11 taken through the riser bore.

FIG. 12 is an end view of the drain cap of FIGS. 10-12.

FIG. 13 is a sectional elevation view of an assembled bioreactor case ofthe present disclosure with a bifurcated fluid path connectable to avessel (not shown) containing fluid to be conditioned using thebioreactor and connectable to collector vessel (not shown) to receivefluid from the bioreactor.

FIG. 14 is a side elevation view of bioreactor case of FIG. 13.

FIG. 15 is a perspective view of bioreactor case of FIG. 13.

FIG. 16 is an exploded view of one embodiment of a cell preparationsystem according to the present disclosure.

FIG. 17 is a perspective assembly view of the cell preparation system ofFIG. 16.

FIG. 18 is a first perspective view of a cell preparation systemcomprising a plurality of modular cell preparation apparatus.

FIG. 19 is a second perspective view of the cell preparation system ofFIG. 18.

FIG. 20 graphs demonstrate that transcriptional induction is robust forCOX2, TSG6, HMOX1, and IL1RN in fluid shear stressed human MSCs. hBMMSC, human bone marrow MSC (first four bars in each graph); hAF MSC,amniotic fluid MSC (second to last bar in each graph); hAD MSC,adipose-derived MSC (last bar in each graph). P-values calculated bypaired t-test, equal variance.

FIG. 21 illustrates representative Western blots showing increasedintracellular COX2 protein, which is reduced by NF-kB antagonistBAY11-7085 (10 μM).

FIG. 22 illustrates TNF-α cytokine suppression assays that highlightfunctional enhancement of MSC immunomodulation. Human MSCspreconditioned by mechanical force (15 dyne/cm2 shear stress for 3 hrs)are placed in static co-culture with lipopoly-saccharide (LPS) orphytohaemagglutinin (PHA)-activated splenocytes including macrophages,neutrophils, NK, B, and T cells. Results of the assays show that TNF-αsecretion by splenocytes is reduced by 10-50% when MSCs are transientlyconditioned with shear stress. Lower values correspond to greateranti-inflammatory potency. hBM MSC, human bone marrow MSC; hAF MSC,amniotic fluid MSC (indicated by a “*” in graph); hAD MSC,adipose-derived MSC (indicated by a “{circumflex over ( )}” in graph).P-values calculated by paired t-test, equal variance.

FIG. 23 illustrates inhibition of COX2 (Indomethacin, 10 μM; NS-398, 10μM) and NF-kB (BAY11-7085, 10 μM) abrogate the positive effects of shearstress, whereas ectopic dmPGE2 (10 μM) mimics MSC suppression. Asterisksindicate p<0.001 compared to Static Vehicle. n=6 indicates six differentMSC donor lines included in data shown.

FIG. 24 illustrates that priming agents complement shear-inducedanti-inflammatory signaling. Darkened bars represent treatmentcombinations that indicate a substantial induction of the pathway byshear stress and cytokines (IFN-γ, 20 ng/ml; TNF-α, 50 ng/ml). In thislow-performance human amniotic MSC line, TSG6 was induced by shearstress only. IDO was induced by IFN-γ only. hAF MSC, human amnioticfluid MSC.

FIGS. 25A-D illustrate that disruption in cellular composition of bonemarrow caused by traumatic brain injury in rats is abrogated byintravenous administration of WSS-preconditioned human MSCs. (A)Immunodetection of endogenous rat MSCs in the bone marrow 24 hours aftertraumatic brain injury by CCI reveals depletion of the CD105⁺ population(counterstain is derived from nuclear fast red). (B) Fewer CD105⁺ MSCsare present in the bone marrow following CCI. (C) CD105 is detected at72 hours after injury (hematoxylin counterstain). (D) Quantification ofCD105 MSC frequency at 72 hours after CCI reveals protection of bonemarrow cellular composition by MSC therapy when administeredintravenously at 24 hours post-CCI. The protective effects on the CD105⁺MSC population are enhanced by WSS preconditioning of MSCs usedtherapeutically (One-way ANOVA, ** *p<0.001).

DETAILED DESCRIPTION

When dealing with multipotent stem cells, such as mesenchymal stem cells(MSCs), controlling the outcome of differentiation is a critical step indeveloping cellular therapies. Generating consistent results requiresprecise control of the cell's environment, which includes a number ofparameters. These include chemical factors such as nutrients, wasteproducts, pH and dissolved gases. Many state of the art bioreactors havebeen designed and built to control and monitor these chemicalparameters.

However, the mechanical environment also plays a significant role in theoutcome of a stem cell. The mechanical environment is dependent upon twomajor elements: the substrate and the dynamic state of the surroundingmedia. Substrate stiffness can be controlled through careful selectionof surface treatments or coatings, which can influence both theadherence of the cells to the substrate as well as differentiationoutcome.

After a population of cells has adhered to a substrate, they aresubjected to mechanical forces applied by the media in which the cellsare immersed. In conventional cell culture, these forces are essentiallyzero since there is no bulk flow of media. Existing bioreactor systemshave constant or variable fluid flow (media), but lack design control ofthe pattern of flow (laminar-uni or bi-directional vs. turbulent) or thedegree of shear forces that interact with the cells. Moving fluid exertson cells a shear stress that is proportional to the fluid velocity andviscosity. Most mechanotransduction studies have been performed in thelaminar flow regime where shear stress behavior is well known andcharacterized by simple equations. Laminar flow is characterized by anon-uniform velocity profile across the cross-section of the flowchannel. Fluid velocity at the boundaries (e.g. walls) can be assumed tobe zero, known as the “no-slip condition” or “boundary condition.” Astraightforward equation describing shear stress (τ) for Newtonianfluids in laminar flow is τ=−μ (du/dy), where μ is viscosity and u isvelocity of the fluid at a particular depth in the channel. Knowingmedia viscosity and fluid velocity, applied shear stress can becalculated.

Cell populations that retain the ability to differentiate into multiplecell types (e.g., stem cells) have proven useful for developing largenumbers of lineage specific differentiated cell populations. Mesenchymalstem cells (MSCs) are one such type of stem cell and are known for beingboth multipotent and self-renewing. MSCs have thus emerged as candidatecellular therapeutics and can potentially provide a sustained source ofbioactive immunomodulatory molecules. However, presently one of theobstacles limiting clinical efficacy of use of MSC is that the inductionof MSC function is heavily dependent upon the presence of cytokines andsignals produced by activated immune cells that in turn initiate theimmunomodulatory activities of MSC. Prior to the present methods, forexample, this variability has translated into unpredictable therapeuticactivity of stem cell compositions.

In some aspects, a bioreactor system described herein provides increasednumbers of stem cells (e.g., MSCs), which have also been predictably andreliably conditioned in vitro with regards to immunomodulatory function.Stem cells, thus obtained, provide therapeutically effective numbers ofMSC which are reliably and consistently conditioned with regards toimmunomodulatory function. In other embodiments, the system provides asource of secreted factors which can be isolated and purified for use astherapeutics.

In some embodiments, such methods can provide conditioned cells, such asMSC, for use in therapy and, for example, suppression of chronic oracute inflammation associated with injury (e.g., neurological injury),graft-versus-host disease, and autoimmunity. Such conditionedcomposition have been demonstrated herein to provide enhance engraftmentpotential for cells upon administration, which could significantlyenhance the efficacy of therapeutic methods.

Certain embodiments include a system for use in producing increasednumbers of therapeutically predictably conditioned cells such as, butnot limited to, MSCs for use in cell-based therapies. In someembodiments, the system described can be used for, among other things,conditioning MSCs to exhibit anti-inflammatory and immunomodulatoryproperties, to treat many types of musculoskeletal trauma andinflammatory conditions when, for example, such conditioned cells orfactors they produce are injected at the site of an injury. In someembodiments, this system comprises a modular bioreactor system thatintegrates control of the hydrodynamic microenvironment to directmechanical-based conditioning of cells, such as, but not limited to MSC.

A bioreactor case is used to condition cells within a stream of mediafluid in accordance with the present invention (e.g., the media ispassed over adherent cells to provide an applied shear force). Anembodiment of a bioreactor case is illustrated in the appended figures,which are discussed below.

FIG. 1 is a plan view of a feed cap 12 of an embodiment of a bioreactorcase. The feed cap 12 comprises a first end 15 and a second end 18 and awall 13 therebetween. The feed cap 12 further comprises a feed passage16 (in dotted lines) proximal to the first end 15 and originating at aninlet fluid connector 11 and terminating at a riser bore 17 extendingfrom the feed passage 16 towards the viewer of FIG. 1.A short portion 19of the feed passage 16 in the embodiment of the feed cap 12 of FIG. 1 isturned perpendicular to the feed passage 16 to connect to the riser bore17. The feed cap 12 includes a sealing face 14 surrounding the wall 13.It should be noticed that there is no second riser bore on the feed cap12 at location 50. The reason for noting this absence will become clearafter consideration of the disclosure that follows.

FIG. 2 is a side elevation view of the feed cap 12 of FIG. 1 revealing aseal 16 inside the inlet fluid connector 11 to sealably engage a fluidconduit (not shown) connected to a pressurized source of a fluid. Thearrow indicates the location of the riser bore 17 proximal to the firstend 15 of the feed cap 12. The wall 13 of the feed cap 12 is illustratedin FIGS. 2 and 3 as being on a top side of the feed cap 12 opposite tothe exterior 20 of the feed cap 12 illustrated as being on a bottom sideof the feed cap 12. It will be understood that the caps and modules ofembodiments of the apparatus of the present invention may be otherwiseoriented without impairing the function.

FIG. 3 is a sectional view of the feed cap 12 of FIGS. 1 and 2 takenthrough the riser bore 17 proximal to the first end 15 of the feed cap12. The riser bore 17 receives fluid from the feed passage 16 anddelivers the fluid flow to one or more intermediate modules (not shownin FIG. 3) at least one of which is sealably received against thesealing face 14.

FIG. 4 is an end view of the feed cap 12 of FIGS. 1-3. FIG. 4illustrates the location of the inlet fluid connector 11 proximal to thefirst end 15 of the feed cap 12, the feed passage 16 extending from theinlet fluid connector 11 to the riser bore 17. It should be noted thatthe inlet fluid connector 11, the feed passage 16 and the riser bore 17of the feed cap 12 are disposed proximal to the first end 15 of the feedcap 12, and that no corresponding fluid passages are disposed proximalto the second end 18 of the feed cap 12.

FIG. 5 is a plan view of an intermediate module 30 of the bioreactorcase of the present invention adapted for sealably and operativelyengaging the feed cap 12. The intermediate module 30 comprises a firstside 31 and a second side 32 (not shown in FIG. 5), each having asealing face 44 to engage the sealing face 14 of the feed cap 12. Theintermediate module 30 further includes a first module bore 34 throughthe intermediate module 30 and proximal to a first end 47 of theintermediate module 30. The module bore 34 is positioned to meet and tocoincide with the riser bore 17 of the feed cap 12 of FIGS. 1-4 uponsealable engagement between the sealing face 44 on the first side 31 ofthe intermediate module 30 and the corresponding sealing face 14 of thefeed cap 12. The intermediate module 30 further includes a second modulebore 134 proximal to a second end 48 of the intermediate module 30 andin a position to coincide with and engage a second feed cap 12 (thesecond feed cap 12 may be identical to the first feed cap 12 but rotated180 degrees about the axis 60 of FIG. 1. It will be understood that thesecond feed cap 12 will engage a sealing face 44 on a second side 32 ofan intermediate module 30 and the first feed cap 12 will engage asealing face 44 of the first side 31 of the intermediate module 30. Itwill further be understood that the absence of a second riser bore 17proximal to the second end 18 of the feed cap 12 enables the sealingface 14 of the feed cap 12 to seal off the second module bore 134 of theintermediate module 30 that is proximal to the second end 48 of theintermediate module 30. The feed cap 12 may, in one embodiment of theapparatus of the present invention, be thus structured to function, in afirst mode in which a feed cap 12 engages the first side 31 of theintermediate module 30, to receive a flow of fluid and deliver the flowto a first end 47 of the intermediate module 30 and, in an invertedsecond mode in which the second feed cap 112 engages the second side 32of the intermediate module 30, to receive a flow of fluid from aplurality of fluid chambers including at least a first fluid chamber 38Aformed between the wall 13 of the first feed cap 12 and the barrier 39of the intermediate module 30 upon engagement of the sealing face 14 ofthe first feed cap 12 with the sealing face 44 of the inverted secondfeed cap 112, and a second fluid chamber 38B formed between the wall 113of the inverted second feed cap 112 and the barrier 38 of theintermediate module 30 upon engagement of the sealing face 14 of thesecond feed cap 112 with the sealing face 114 of the inverted secondfeed cap 112. The structure of the second feed cap 112 is discussedfurther in connection with FIGS. 9-12.

The intermediate module 30 of FIG. 5 further includes a diffuser feedchannel 35 proximal to the first end 47 of the intermediate module 30and originating at the module bore 34 and terminating at a distributionchannel 36. The diffuser feed channel 35 extends generally distal to thefirst module bore 34 proximal to the first end 47 of the intermediatemodule 30 to the distribution channel 36. The distribution channel 36 ofthe intermediate module 30 receives a fluid flow from the diffuser feedchannel 35 and extends from the diffuser channel 35 towards a first edge57 of the intermediate module 30 and also from the diffuser channel 35towards a second edge 58 of the intermediate module 30. Similarly, theintermediate module 30 of FIG. 5 further includes an infuser drainchannel 135 proximal to the second end 48 of the intermediate module 30and originating at the infusers 133 and terminating at the gatheringchannel 136. The infuser drain channel 135 extends generally proximal tothe second module bore 134 proximal to the second end 48 of theintermediate module 30 to the gathering channel 136. The gatheringchannel 136 of the intermediate module 30 receives a fluid flow from theinfusers 133 and directs the fluid flow to the second module bore 134through the infuser drain channel 135.

FIG. 6 is a side elevation view of the intermediate module 30 of FIG. 5revealing the positioning of a plurality of fluid diffusers 33 disposedwithin the first fluid chamber 38A, a plurality of fluid diffusers 33disposed within the second fluid chamber 38B, a plurality of fluidinfusers 133 disposed within the first fluid chamber 38A and a pluralityof fluid infusers 133 disposed within the second fluid chamber 38B, thepluralities of fluid diffusers 33 being proximal to a first end 47 ofthe intermediate module 30 and the pluralities of fluid infusers 133being proximal to the second end 48 of the intermediate module 30. Itwill be understood that the term “diffuser,” as used herein, means adevice for reducing the velocity and increasing the static pressure of afluid. It will be understood that the term “infuser,” as used herein,means a device for receiving a low velocity, high pressure flow andconverting into a higher velocity, lower pressure flow.

FIG. 7 is an enlarged view of a plurality of connected fluid passagesand infusers 133 at a second end 48 of the side elevation view of theintermediate module 30 of FIG. 6. It will be understood that thearrangement of fluid passages in the embodiment of the apparatus of thepresent invention illustrated in the appended figures can be arranged indifferent ways without materially altering their function. FIG. 7reveals an intermediate module 30 having a second module bore 34therethrough. The second module bore 134 is fluidically connected to agathering channel 136 through an infuser drain channel 135. Thegathering channel 136 extends into the page and out of the page, as seenby the viewer of FIG. 7, to fluidically connect with a plurality ofdiffusers 133 through a corresponding plurality of port channels 137.The fluid enters the first fluid chamber 38A and the second fluidchamber 38B which are separated one from the other by the barrier 39, tothe infusers 133, to the port channels 137, the gathering channel 136,the infuser drain channel 135 to the second module bore 134. It will benoted that FIG. 7 illustrates a portion 40 of the plurality of portchannels 137 extend into the first and second fluid chambers 38A and38B.

FIG. 8 is a sectional end view of the intermediate module of FIGS. 5-7revealing the locations of the infusers 133 that receive flow from thefirst flow chamber 38A and the second flow chamber 38B. It will beunderstood that FIG. 8 may also be viewed as a sectional end view of theintermediate module 30 revealing the locations of the diffusers 33 thatintroduce flow into the first flow chamber 38A and the second flowchamber 38B. FIG. 8 illustrates the spacing of the infusers 133 (at thesecond end 48 of the intermediate module 30) and of the diffusers 33 (atthe first end 47 of the intermediate module 30).

FIG. 9 is a plan view of a drain cap 112 of an embodiment of abioreactor case of the present invention. The drain cap 112 comprises afirst end 115 and a second end 118 and a wall 113 therebetween. Thedrain cap 112 further comprises a drain passage 116 (in dotted lines)proximal to the first end 115 and originating at a riser bore 117 andterminating at an outlet fluid connector 111. The riser bore 117 extendsfrom the drain passage 116 towards the viewer of FIG. 9. A short portion119 of the drain passage 116 in the embodiment of the drain cap 112 ofFIG. 9 is turned perpendicular to the drain passage 116 to connect tothe riser bore 117. The drain cap 112 includes a sealing face 114surrounding the wall 113.

FIG. 10 is a side elevation view of the drain cap 112 of FIG. 9revealing a seal 116 inside the outlet fluid connector 111 to sealablyengage a fluid conduit (not shown) connected to a collector to receive aconditioned fluid from the bioreactor 10. The arrow indicates thelocation of the riser bore 117. The wall 113 of the drain cap 112 isillustrated as being on a top side of the drain cap 112 opposite to theexterior 120 of the drain cap 112 illustrated as being on a bottom sideof the drain cap 112. It will be understood that the caps and modules ofembodiments of the apparatus of the present invention may be otherwiseoriented without impairing the function.

FIG. 11 is a sectional side view of the drain cap 112 of FIGS. 9 and 10taken through the riser bore 117. The riser bore 117 receives fluid fromthe first fluid chamber 38A and the second fluid chamber 38B by way ofthe second module bore 134 of the intermediate module 30 (see FIGS. 5-8)and delivers the flow to the drain cap 112 having a sealing surface 114sealably received against the sealing face 44 of the first side 31 ofthe intermediate module 30.

FIG. 12 is a sectional end view of the drain cap 112 of FIGS. 9-11. FIG.12 illustrates the location of the outlet fluid connector 111 proximalto the first end 115 of the drain cap 112, and the drain passage 116extending from the outlet fluid connector 111 to the riser bore 117. Itshould be noted that the outlet fluid connector 111, the drain passage116 and the riser bore 117 of the drain cap 112 are disposed proximal tothe first end 115 of the drain cap 112, and that no corresponding fluidpassages are disposed proximal to the second end 118.

FIG. 13 is a perspective view of an assembled bioreactor case 10 of thepresent invention with the inlet fluid connector 11 and the outlet fluidconnector 111 of the feed cap 12 and the drain cap 112, respectively,omitted from the FIG. 13 to better reveal the positioning of the riserbore 17 of the feed cap 12, the riser bore 117 of the drain cap 112, thefirst module bore connectable to a vessel (not shown) containing fluidto be used in conditioning stem cells disposed within the flow chambers38A and 38B of the bioreactor 10 and an outlet fluid connector 111 (notshown) connectable to a vessel to receive conditioned fluid from thebioreactor.

It will be understood that the assembled bioreactor case 10 illustratedin FIG. 13 comprises a feed cap 12, a drain cap 112 and an intermediatemodule 30 therebetween. It will be further understood that the rate anddirection of flow of a fluid through the bioreactor case 10 depends onthe pressure differential (difference between the pressure at the fluidinlet connector 11 and the pressure at the fluid outlet connector 111),the resistance to fluid flow through the bioreactor 10, the viscosity ofthe fluid and other factors. The feed cap 12, the intermediate module 30and the drain cap 112 may be secured in the assembled configuration ofthe bioreactor case 10 of FIG. 13 in various ways including the use ofclamps, bands, ties and the like.

Alternately, the feed cap 12, the intermediate module 30 and the draincap 112 may be secured in the assembled configuration of the bioreactorcase 10 of FIG. 13 by providing a feed cap 12, a drain cap 112 and anintermediate module 30 having embedded or connected magnetic membersoriented for mutual attraction. FIGS. 14 and 15 illustrate an embodimentof the bioreactor case 10 having a plurality of rare earth magnets 49embedded at a plurality of positions along the perimeter of the feed cap12 and a corresponding plurality of rare earth magnets may be embeddedin aligned positions along the perimeter of the drain cap 112.

Referring now to FIGS. 16 and 17, another embodiment of a bioreactorcase 200 is shown in perspective and exploded views, respectively. Forpurposes of clarity, not all elements are labeled with reference numbersin each figure. Bioreactor case 200 comprises an inlet fluid feed plate212, a plurality of intermediate module plates 232, and a base moduleplate 234. Each intermediate module plate 232 comprises a first end 233,a second end 235, a first side 273, and a second side 275. In addition,intermediate module plates 232 are vertically stacked between inletfluid feed plate 212 and base module plate 234.

As shown in FIGS. 18 and 19, bioreactor case 200 can be assembled as acomponent in a modular cell preparation apparatus 300. In the embodimentshown in FIGS. 18 and 19, a plurality of modular cell preparationapparatus 300 are assembled as components in a cell preparation system400. Cell preparation system 400 comprises a base 450 configured tosupport multiple modular cell preparation apparatus 300. In theembodiment shown, each cell preparation system comprises bioreactor case200, reservoir 220, first housing 271, second housing 272 and pump 250.Reservoir 220 is in fluid communication with bioreactor case 200 viasupply conduit 215 and return conduit 225.

Referring specifically now to FIGS. 16 and 17, bioreactor case 200comprises inlet fluid feed plate 212 with a central channel 217 having afirst fluid inlet 211 and a first fluid outlet 221. Inlet fluid feedplate 212 also comprises a second fluid inlet 231 and a second fluidoutlet 241. As shown in FIGS. 18 and 19, first fluid inlet 211 andsecond fluid outlet 241 may be coupled to reservoir 220, while firstfluid outlet 221 and second fluid inlet 231 may be coupled to a pump250. In certain embodiments, pump 250 may be configured as a roller(e.g. peristaltic) pump. Pump 250 comprises a rolling element 252 and acompressible conduit 254. During operation rolling element 252 isrotated by an electric motor 260. Fluid from reservoir 220 is drawnthrough conduit 215 into first fluid inlet 211, through central channel217 and first fluid outlet 221 into compressible conduit 254. Fluid isthen directed to second fluid inlet 231 and to a distribution port 238in fluid communication with intermediate module plates 232.

Intermediate module plates 232 each comprise an inlet port 237 in fluidcommunication with distribution port 238. Each intermediate module plate232 further comprises a distribution channel 253 in fluid communicationwith inlet port 237. Each distribution channel 253 and inlet port 237are proximal to first end 233 of intermediate module plate 232. Eachdistribution channel 253 extends across a culture plate 239 anddistributes fluid over culture plate 239. After flowing across cultureplate 239, fluid is collected by gathering channel 263, which is influid communication with a return port 261 in each intermediate moduleplate 232. Gathering channel 263 and return port 261 are proximal tosecond end 235 of intermediate module plate 232. Accordingly, fluid mustflow across culture plate 239 in order to enter gathering channel 263.Return ports 261 are further in fluid communication with second fluidoutlet 241, which is in fluid communication with reservoir 220 viareturn conduit 225.

The flow of fluid culture plates 239 from distribution channels 253 togathering channels 263 subjects cells on culture plates 239 to a shearstress. The ability to recycle fluid from reservoir 220 and throughbioreactor case 200 (including multiple intermediate module plates 232and culture plates 239) via pump 250 allows the shear stress to beapplied for extended (theoretically unlimited) periods of time.

In exemplary embodiments, it is desirable to control the amount of shearstress applied to cells on culture plates 239 at a uniform level and tominimize the differences in applied shear stress at different locationson culture plates 239. One factor in maintaining a uniform applied shearstress to the cells is the velocity of the fluid media relative to thecells and culture plate 239. Reducing variations in the fluid velocitycan help to maintain a more uniform applied shear stress. As shown inFIG. 16, distribution channels 253 and gathering channels 263 extendsubstantially across a width W of the surface of intermediate moduleplate 232 (e.g. the dimension between first side 273 and second side 275of intermediate module plate 232). In certain embodiments, the length Lof distribution channels 253 and gathering channels 263 (e.g. thedistance between each end of the channels measured parallel to dimensionW of intermediate module plate 232) extend across a majority of width Wof intermediate module plate 232. In particular embodiments, length L isat least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, or95 percent of width W. This configuration can provide a more uniformvelocity of fluid media across culture plates 239 as compared to, forexample, inlet and outlet ports that do not extend across a majority ofculture plates 239. Cells adhered to culture plates 239 will in turn besubjected to a more uniform shear stress.

The level of shear stress applied to the cells can also be controlled byadjusting various operational parameters. For example, the pressureapplied to the fluid via pump 250 can be altered by adjusting the amountthat rolling element 252 compresses compressible conduit 254. The volumeand velocity of fluid flowing across culture plates 239 can also beadjusted by varying the speed of electric motor 260, which in turn willalter the rotational speed of rolling element 252. Additional controlcan be accomplished by selection of specific dimensions and geometriesfor components including, but not limited to the surface finish (e.g.roughness), length and width of culture plates 239, as well as thedistance between intermediate module plates 232.

In addition, the use of a plurality of intermediate module plates 232with culture plates 239 in a vertical stack allows for an increasednumber of cells to be subjected to shear stress. Furthermore, the use ofmultiple modular cell preparation apparatus 300 operating in parallel incell preparation system 400 can further increase the number of cellssubjected to shear stress in preparation for further processing oranalysis.

In certain embodiments, modular cell preparation apparatus 300(operating individually or as components in cell preparation system 400)can be operated to control the hydrodynamic microenvironment to directmechanotransduction conditioning of cells such as, but not limited toMSC, in a controlled manner. By way of example, the studies providedherein demonstrate that the methods and apparatus described conditioncell populations, including for example MSC, by subjecting them to auniform and controllable shear stress as needed to condition such cellsto express a particular activity, including, but not limited to, theinduction and release of immunomodulatory factors.

The studies provided in the examples below demonstrate methods thathuman cell cultures, for example MSC, subject to shear stress of thetype similar to that provided by the present apparatus, albeit using aless flexible system, using a device that is far more limited and lesspreparative in the scale of its abilities to administer shear stress.However, these studies illustrate that shear stress can be used tocondition cells to express a particular activity, such as, but notlimited to the induction and release of immunomodulatory factors.

The results presented herein demonstrate that functional MSCs can bedirectly conditioned to express and produce anti-inflammatory andimmunomodulatory factors. In the context of cellular therapy, thistechnique promises to provide relief to patients affected by or at riskfor inflammation associated with injury or disease. This indicates thatconditioning of MSCs using shear stress of the type provided by thepresent system substantially increases their ability to inhibitinflammatory cells in pre-existing inflammatory environments and may aidin the prevention and resolution of inflammation.

Moreover, by using the system described herein, conditioning can becompleted more rapidly, uniformly and reliably than when usingalternatively available techniques of inducing MSC cell immunomodulatoryactivity, including for example, the production of anti-inflammatorymolecules. The system described to condition cells can be particularlyadvantageous when a subject's own (autologous) cells are to be used as atherapeutic and a method for cell expansion and conditioning isrequired.

In the absence of conditioning, naive MSCs express little to none of thekey mediators of immunosuppression such as the multifunctionalanti-inflammatory proteins TNF-α stimulated protein 6 (TSG-6),prostaglandin E2 (PGE2), and interleukin (IL)-1 receptor antagonist(IL1RN). As detailed in the examples below, MSCs derived from threehuman tissue sources, bone marrow, adipose, and amniotic fluid, were allfound to be responsive to this system of shear stress-based conditioningsuch that activation of immunomodulatory signaling was detectable tovarying extents. Specifically, evaluation of conditioned human bonemarrow-derived MSCs, using laminar shear stress of the type provided bythe present system stimulated profound up-regulation of gene expressionwith from 6- to 120-fold increases, in the transcription of MSC genesencoding TSG-6, COX-2, IL1Ra, HMOX-1, LIF, and KLF2.

Exemplary embodiments include methods for providing a population ofconditioned cells, the method comprising: obtaining a population ofcells and subjecting the cells to a controlled shear stress. Certainembodiments include methods for providing a population of conditionedcells, the method comprising: obtaining a population of cells; culturingsaid cells in a cell media; and subjecting the cells to a controllableshear stress of sufficient force to condition the cells. In someembodiments, the cells are originally obtained from a mammal. In someembodiments, the cells are originally obtained from a companion animal.In preferred embodiments, the cells are originally obtained from ahuman. In some embodiments, the cells are originally obtained from bonemarrow. In some embodiments, the cells are originally obtained fromamniotic fluid while in other embodiments. While in some embodiments,the cells are originally obtained from adipose tissue. In someembodiments, the cells subjected to a controlled shear stress are MSC.

In additional embodiments, are methods of obtaining a therapeuticallyeffective number of conditioned cells. In some embodiments, are methodsof obtaining a therapeutically effective number of cells conditionedusing the apparatus and methods described herein. In some embodiments,are methods of obtaining a therapeutically effective number of cellsconditioned using the method comprising: obtaining a population ofcells; applying a controlled shear stress of sufficient force tocondition such cells to act as desired. In some other embodiments, aremethods of obtaining a therapeutically effective number of cellsconditioned using the method comprising: obtaining a population ofcells; applying a controlled shear stress of sufficient force tocondition such cells to act as desired. In some embodiments are methodsof obtaining a therapeutically effective number of cells conditionedusing the method comprising: obtaining a population of cells; culturingthe cells on a first culture surface in a cell media, such that thecells adhere to on the first culture surface; and applying a controlledshear stress of sufficient force to condition such cells to act asdesired. In some embodiments are methods of obtaining a therapeuticallyeffective number of cells conditioned using the method comprising:obtaining a population of cells; culturing the cells on a culturesurface in a cell media, such that the cells adhere to on the barrier;and applying a controlled shear stress of sufficient force to conditionsuch cells to act as desired.

In similar embodiments, are methods for providing a population ofconditioned cells comprising: obtaining a population of cells; culturingthe cells in a culture system in a cell media, such that the cellsadhere to the barrier; passing the cell media over said cells to providea controlled fluid laminar shear stress of sufficient force to conditionsaid cells. In some embodiments, the conditioned cells expressanti-inflammatory activity. In some embodiments, the anti-inflammatoryactivity includes increased expression of genes selected from a groupcomprising those that encode TSG-6, COX-2, IL1RN, HMOX-1, LIF, or KLF2.In some embodiments, the activity includes increased expression of COX2protein by the conditioned cells.

Additional embodiments include compositions comprising cells that havebeen conditioned using controlled shear stress in the apparatusdescribed in claims 1-10. Some embodiments include compositionscomprising cells that have been conditioned using a method comprising:obtaining a population of cells; culturing said cells in a culturesystem in a cell media, such that the cells adhere to the barrier; andapplying a fluid laminar shear stress of sufficient force to conditionsaid cells. In similar embodiments, are compositions comprising cellsthat have been conditioned using a method for providing a population ofconditioned cells comprising: obtaining a population of cells; culturingthe cells in a culture system in a cell media, such that the cellsadhere to the barrier; passing said cell media over said cells toprovide a fluid laminar shear stress of sufficient force to conditionsaid cells. In some embodiments, the compositions comprise conditionedcells that express anti-inflammatory activity. In some embodiments, theanti-inflammatory activity includes increased expression of genesselected from a group comprising those that encode TSG-6, COX-2, IL1RN,HMOX-1, LIF, or KLF2. In some embodiments, the compositions compriseconditioned cells that express increased levels of COX2 protein. Inadditional embodiments the described device and methods may be used tostimulate the expression and release of anti-inflammatory factors, whichcan be isolated from the media and be used as therapeutics.

In additional embodiments are methods of treating a subject in need ofsuch a treatment with cells conditioned by the methods described. Inalternative embodiments are methods of treating a subject in need ofsuch a treatment may include factors released by cells conditioned usingthe described methods.

In some embodiments, methods of treating a subject include but are notlimited to, obtaining a population of conditioned cells produced inaccordance with the described system and administering the cells to thesubject in need of treatment. In some embodiments, the subject is inneed of an anti-inflammatory therapy and the population of cells arehuman MSC whose anti-inflammatory activity has been induced using thedescribed system. In some embodiments, a therapeutic dose of such cellsmay comprise at least 1×10², 1×10³, 1×10⁴, 1×10⁵ or 1×10⁶ cells whichare introduced into the subject in need of therapy. In some embodimentsanti-inflammatory activity of the conditioned cell population can beused to treat acute disorders such as, but not limited to, muscularskeletal injuries such as orthopedic or spinal cord injury or traumaticbrain injury.

Cell culture conditioning systems are described in various embodimentsherein and it is appreciated that additional methods for the culture andmaintenance of cells, as would be known to one of skill, may be usedwith the present embodiments. In certain embodiments, for culture,various matrix components may be used in culturing, maintaining, ordifferentiating human stem cells. In addition to those described in theexamples below, for example, collagen IV, fibronectin, laminin, andvitronectin in combination may be used to coat a culturing surface as ameans of providing a solid support for pluripotent cell growth.Matrigel™ may also be used to provide a substrate for cell culture andmaintenance of human pluripotent stem cells. Matrigel™ is a gelatinousprotein mixture secreted by mouse tumor cells and is commerciallyavailable from BD Biosciences (New Jersey, USA). This mixture resemblesthe complex extracellular environment found in many tissues and is usedby cell biologists as a substrate for cell culture.

In some embodiments of cell culturing, once a culture container is full(e.g., confluent), the colony is split into aggregated cells or evensingle cells by any method suitable for dissociation, which cells arethen placed into new culture containers for passaging. Cell passaging orsplitting is a technique that enables cells to survive and grow undercultured conditions for extended periods of time. Cells typically wouldbe passaged when they are about 70%-100% confluent.

In certain aspects, starting cells for the present conditioning systemmay comprise at least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ cells or any range derivable therein. The starting cellpopulation may have a seeding density of at least or about 10, 10¹, 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ cells/mL, or any range derivable therein.

As the basal medium, in addition to that described in the examplesbelow, a range of media is available including defined medium, such asEagle's Basal Medium (BME), BGJb, CMRL 1066, Glasgow MEM, Improved MEMZinc Option, Iscove's modified Dulbecco's medium (IMDM), Medium 199,Eagle MEM, αMEM, DMEM, Ham, RPMI 1640, and Fischer's media. Additionalexamples of media that may be used according to the embodiments include,without limitation, Lonza Therapeak (chemically defined) medium, IrvineScientific Prime-XV (SFM or XSFM), PromoCell MSC Growth Medium (DXF),StemCell Technologies Mesencult (ACF), or human platelet orplatelet-lysate enriched medium.

In further embodiments, the media can also contain supplements such asB-27 supplement, an insulin, transferrin, and selenium (ITS) supplement,L-Glutamine, NEAA (non-essential amino acids), P/S(penicillin/streptomycin), N2 supplement (5 μg/mL insulin, 100 μg/mLtransferrin, 20 nM progesterone, 30 nM selenium, 100 μM putrescine andβ-mercaptoethanol (β-ME). It is contemplated that additional factors mayor may not be added, including, but not limited to fibronectin, laminin,heparin, heparin sulfate, retinoic acid.

Additional factors may be added to a media for use in conjunction withsheer stress for generating a conditioned composition, such as apopulation of conditioned cells. Thus, in some embodiments, at least onechemical modulator of hematopoiesis may be applied before, during, orafter biomechanical stimulation. Examples of additional components thatcould be added to media include, without limitation, Atenolol, Digoxin,Doxazosin, Doxycycline, Fendiline, Hydralazine,13-hydroxyoctadecadienoic acid (13(s)-HODE), Lanatoside C,NG-monomethyl-L-arginine (L-NMMA), Metoprolol, Nerifolin, Nicardipine,Nifedipine, Nitric oxide (NO) or NO signaling pathway agonists,1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), Peruvoside,Pindolol, Pronethalol, Synaptosomal protein (SNAP), SodiumNitroprusside, Strophanthidin, Todralazine, 1,5-Pentamethylenetetrazole,Prostaglandin E 2 (PGE2), PGE2 methyl ester, PGE2 serinol amide,11-deoxy-16,16-dimethyl PGE2, 15(R)-15-methyl PGE2, 15 (S)-15-methylPGE2, 6,16-dimethyl PGE2, 16,16-dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester, 16-phenyl tetranor PGE2, 19(R)-hydroxy PGE2, ProstaglandinB2, Prostacyclin (PGI2, epoprostenol), 4-Aminopyridine, 8-bromo-cAMP,9-deoxy-9-methylene PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, aPGE2 receptor agonist, Bapta-AM, Benfotiamine, Bicuclline,(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), Bradykinin, Butaprost,CaylO397, Chlorotrianisene, Chlorpropamide, Diazoxide, EicosatrienoicAcid, Epoxyeicosatrienoic Acid, Flurandrenolide, Forskolin, Gaboxadol,Gallamine, Indanyloxyacetic acid 94 (IAA 94), Imipramine, KynurenicAcid, L-Arginine, Linoleic Acid, LY171883, Mead Acid, Mebeverine, 12Methoxydodecenoic acid, N-Formyl-Met-Leu-Phe, Prostaglandin E2 receptorEP2-selective agonist (ONO-AEl-259), Peruvoside, Pimozide, Pindolol,Sodium Nitroprusside, Sodium Vanadate, Strophanthidin, Sulprostone,Thiabendazole, Vesamicol, 1,2-Didecanoyl-glycerol (10:0), 11,12Epoxyeicosatrienoic acid,l-Hexadecyl-2-arachidonoyl-glycerol,5-Hydroxydecanoate, 6-Formylindolo [3,2-B] carbazole, Anandamide (20:3,n-6), Carbacyclin, Carbamyl-Platelet-activating factor (C-PAF), orS-Farnesyl-L-cysteine methyl ester.

In further aspects, a media can include one or more growth factors, suchas members of the epidermal growth factor family, e.g., EGF, members ofthe fibroblast growth factor family (FGFs) including FGF2 and/or FGF8,members of the platelet derived growth factor family (PDGFs),transforming growth factor (TGF)/bone morphogenetic protein (BMP)/growthand differentiation factor (GDF) factor family antagonists including butnot limited to noggin, follistatin, chordin, gremlin, cerberus/DANfamily proteins, ventropin amnionless, TGF, BMP, and GDF antagonistscould also be added in the form of TGF, BMP, and GDF receptor-Fcchimeras. Other factors that may or may not be added include moleculesthat can activate or inactivate signaling through Notch receptor family,including but not limited to proteins of the Delta-like and Jaggedfamilies as well as gamma secretase inhibitors and other inhibitors ofNotch processing or cleavage such as DAPT. Additional growth factors mayinclude members of the insulin like growth factor family (IGF), thewingless related (WNT) factor family, and the hedgehog factor family.

In still further aspects, a media can include one or more priming agentssuch as an inflammatory cytokine, LPS, PHA, Poly I:C, and/or ConA.Additional priming agents that may be used according the embodimentsinclude those detailed Wagner et al., 2009, which is incorporated hereinby reference. Additional factors may be added particularly in furtherdifferentiation medium to promote cell progenitor proliferation andsurvival as well as self renewal and differentiation, including but notlimited to dimethyl-prostaglandin E2, iloprost, and other similarproducts of arachidonic acid metabolism.

The medium can be a serum-containing or serum-free medium. Theserum-free medium may refer to a medium with no unprocessed orunpurified serum and accordingly, can include media with purifiedblood-derived components or animal tissue-derived components (such asgrowth factors). From the aspect of preventing contamination withheterogeneous animal-derived components, serum can be derived from thesame animal as that of the cell(s).

The medium may contain or may not contain any alternatives to serum. Thealternatives to serum can include materials which appropriately containalbumin (such as lipid-rich albumin, albumin substitutes such asrecombinant albumin, plant starch, dextrans and protein hydrolysates),transferrin (or other iron transporters), fatty acids, insulin, collagenprecursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, orequivalents thereto. The alternatives to serum can be prepared by themethod disclosed in International Publication No. WO98/30679, forexample. Alternatively, any commercially available materials can be usedfor more convenience. The commercially available materials includeknockout Serum Replacement (KSR), Chemically-defined Lipid concentrated(Gibco), and Glutamax (Gibco).

The medium can also contain fatty acids or lipids, amino acids (such asnon-essential amino acids), vitamin(s), growth factors, cytokines,antioxidant substances, 2-mercaptoethanol, pyruvic acid, bufferingagents, and inorganic salts. The concentration of 2-mercaptoethanol canbe, for example, about 0.05 to 1.0 mM, and particularly about 0.1 to0.5, or 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2,2.5, 5, 7.5, 10 mM or any intermediate values, but the concentration isparticularly not limited thereto as long as it is appropriate forculturing the stem cell(s).

The cells may be cultured in a volume of at least or about 0.005, 0.010,0.015, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 800, 1000, 1500 mL, or any range derivabletherein, depending on the needs of the culture. The bioreactor may havea volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75,100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or anyrange derivable therein.

The culture surface and chamber formed between the wall 13 of the feedcap 12 and the barrier 39 of the intermediate module 30 when theapparatus is assembled can be prepared with cellular adhesive or notdepending upon the purpose. The cellular adhesive culture vessel can becoated with a suitable substrate for cell adhesion (e.g. extracellularmatrix [ECM]) to improve the adhesiveness of the vessel surface to thecells. The substrate used for cell adhesion can be any material intendedto attach stem cells or feeder cells (if used). Non-limiting substratesfor cell adhesion include collagen, gelatin, poly-L-lysine,poly-D-lysine, poly-D-ornithine, laminin, vitronectin, and fibronectinand mixtures thereof, for example, protein mixtures fromEngelbreth-Holm-Swarm mouse sarcoma cells (such as Matrigel™ or Geltrex)and lysed cell membrane preparations. In specific embodiments cultureincludes a matrix comprising poly-L-lysine (or poly-D-lysine) andlaminin.

Other culturing conditions can be appropriately defined. For example,the culturing temperature can be about 30 to 40° C., for example, atleast or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C. but particularlynot limited to them. The CO₂ concentration can be about 1 to 10%, forexample, about 2 to 7%, or any range derivable therein. The oxygentension can be at least or about 1, 5, 8, 10, 20%, or any rangederivable therein.

Essentially free of an “externally added” component refers to a mediumthat does not have, or that have essentially none of, the specifiedcomponent from a source other than the cells in the medium. “Essentiallyfree” of externally added growth factors or polypeptides, such as FGF orEGF etc., may mean a minimal amount or an undetectable amount of theexternally added component. For example, a medium or environmentessentially free of FGF or EGF polypeptide can contain less than 1, 0.9,0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, 0.001 ng/mL or any rangederivable therein.

In some embodiments, cells conditioned using the system described have avariety of therapeutic uses. In particular, if the cells are human MSCs,diseases or disorders for which such conditioned cells can be usedtherapeutically, or alternatively those diseases or disorders for whichtherapy with the factors produced and isolated from cultured cells,including but not limited to MSCs subjected to conditioning with thesystem described include, but are not limited to, autoimmune disorders(including but not limited to Rheumatoid Arthritis (RA), Systemic LupusErythematosis (SLE)), graft-versus-host disease, Crohn's disease,inflammatory bowel disease, neurodegenerative disorders, neuronaldysfunctions, disorders of the brain, disorders of the central nervoussystem, disorders of the peripheral nervous system, neurologicalconditions, disorders of memory and learning, cardiac arrhythmias,Parkinson's disease, ocular disorders, spinal cord injury, disordersrequiring neural healing and regeneration, Multiple Sclerosis (MS),Amyelotrophic Lateral Sclerosis (ALS), Parkinson's disease, stroke,chronic or acute injury, bone repair, traumatic brain injury, orthopedicand spinal conditions, cartilage skeletal or muscular disorders,osteoarthritis, osteonecrosis, cardiovascular diseases, blood vesseldamage linked to heart attacks or diseases such as critical limbischemia, peripheral artery disease, atherosclerosis, and thosebenefiting from neovascularization, wounds, burns and ulcers.

In certain embodiments the presently disclosed system can be applied tocondition cells and improve their immune regulatory properties. Incertain embodiments, such compositions can be administered incombination with one or more additional compounds or agents (“additionalactive agents”) for the treatment, management, and/or prevention ofamong other things autoimmune diseases and disorders. Such therapies canbe administered to a patient at therapeutically effective doses to treator ameliorate, among other things, immunoregulatory disease ordisorders.

Toxicity and therapeutic efficacy of such conditioned cell or factorcompositions can be determined by standard pharmaceutical procedures,using for example, cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, expressed as the ratio LD₅₀/ED₅₀. Compositions that exhibit largetherapeutic indices are preferred. Compounds that exhibit toxic sideeffects may be used in certain embodiments, however, care should usuallybe taken to design delivery systems that target such compositionspreferentially to the site of affected tissue, in order to minimizepotential damage to unaffected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosages for use in humans. The dosages of suchcompositions lie preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending on the dosage form employed and the route ofadministration utilized. For any composition, the therapeuticallyeffective dose may be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test composition that achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Plasma levels may bemeasured, for example, by high performance liquid chromatography.

When the therapeutic treatment of among other things autoimmunedisorders is contemplated, the appropriate dosage may also be determinedusing animal studies to determine the maximal tolerable dose, or MTD, ofa bioactive agent per kilogram weight of the test subject. In general,at least one animal species tested is mammalian. Those skilled in theart regularly extrapolate doses for efficacy and avoiding toxicity toother species, including human. Before human studies of efficacy areundertaken, Phase I clinical studies will help establish safe doses.

Additionally, the bioactive agent may be coupled or complexed with avariety of well-established compositions or structures that, forinstance, enhance the stability of the bioactive agent, or otherwiseenhance its pharmacological properties (e.g., increase in vivohalf-life, reduce toxicity, etc.).

Cells conditioned using the present system or factors released from suchcells and other such therapeutic agents can be administered by anynumber of methods known to those of ordinary skill in the art including,but not limited to, cell insertion during surgery, intravenous (I.V.),intraperitoneal (I.P.), intramuscular (I.M.), or intrathecal injection,inhalation, subcutaneous (sub-q), or topically applied (transderm,ointments, creams, salves, eye drops, and the like).

The following examples section provides further details regardingexamples of various embodiments. It should be appreciated by those ofskill in the art that the techniques disclosed in the examples thatfollow represent techniques and/or compositions discovered by theinventors to function well. However, those of skill in the art should,in light of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. These examples are illustrations of the methods andsystems described herein and are not intended to limit the scope of theinvention. Non-limiting examples of such include, but are not limitedto, those presented below.

As used herein, and unless otherwise indicated, the terms “treat,”“treating,” “treatment” and “therapy” contemplate an action that occurswhile a patient is suffering from a disease or disorder that reduces theseverity of one or more symptoms or effects of such disease or disorder.Where the context allows, the terms “treat,” “treating,” and “treatment”also refers to actions taken toward ensuring that individuals atincreased risk of a disease or disorder, are able to receive appropriatesurgical and/or other medical intervention prior to onset of a diseaseor disorder. As used herein, and unless otherwise indicated, the terms“prevent,” “preventing,” and “prevention” contemplate an action thatoccurs before a patient begins to suffer from a disease or disorder,that delays the onset of, and/or inhibits or reduces the severity of adisease or disorder.

As used herein, and unless otherwise indicated, the terms “manage,”“managing,” and “management” encompass preventing, delaying, or reducingthe severity of a recurrence of a disease or disorder in a patient whohas already suffered from such a disease, disorder or condition. Theterms encompass modulating the threshold, development, and/or durationof a disease or disorder or changing how a patient responds to a diseaseor disorder.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of cells, factor or compound is an amount sufficientto provide any therapeutic benefit in the treatment or management of adisease or disorder, or to delay or minimize one or more symptomsassociated with a disease or disorder.A therapeutically effective amountmeans an amount of the cells, factor or compound, alone or incombination with one or more other therapies and/or therapeutic agentsthat provide any therapeutic benefit in the treatment or management of adisease or disorder. The term “therapeutically effective amount” canencompass an amount that alleviates a disease or disorder, improves orreduces a disease or disorder, improves overall therapy, or enhances thetherapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of cells, factor or compound is an amount sufficientto prevent or delay the onset of a disease or disorder, or one or moresymptoms associated with a disease or disorder, or prevents or delaysits recurrence.A prophylactically effective amount of cells, factors orcompound means an amount of the cells, factor or compound, alone or incombination with one or more other treatment and/or prophylactic agentthat provides a prophylactic benefit in the prevention of a disease ordisorder. The term “prophylactically effective amount” can encompass anamount of cells, factor or compound that prevents a disease or disorder,improves overall prophylaxis, or enhances the prophylactic efficacy ofanother prophylactic agent. The “prophylactically effective amount” canbe prescribed prior to, for example, the disease or disorder.

As used herein, “patient” or “subject” includes mammalian organismswhich are capable of suffering from a disease or disorder as describedherein, such as human and non-human mammals, for example, but notlimited to, rodents, mice, rats, non-human primates, companion animalssuch as dogs and cats as well as livestock, e.g., sheep, cow, horse,etc.

As used herein, “MSC” are Mesenchymal Stem Cells, such cells have alsobeen referred to as Mesenchymal Stromal Cells.

As used herein, “controlled shear stress” refers to the ability to setthe amount of shear stress applied to the cells by adjusting the flowrate of media across the surface. The stress is uniformly applied acrossthe entire surface area of the plate.

As used herein, “conditioned cells” refers to cells which expressadditional functionality as a result of having been exposed to a shearstress.

The corresponding structures, materials, acts, and equivalents of allmeans or steps plus function elements in the claims below are intendedto include any structure, material, or act for performing the functionin combination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but it is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated.

Without further elaboration, it is believed that one skilled in the artcan, using the description herein, utilize the present methods to itsfullest extent. The embodiments described herein are to be construed asillustrative and not as constraining the remainder of the disclosure inany way whatsoever. While preferred embodiments have been shown anddescribed, many variations and modifications thereof can be made by oneskilled in the art without departing from the spirit and teachings ofthe presently disclosed methods.

Accordingly, the scope of protection is not limited by the descriptionset out above, but is only limited by the claims, including allequivalents of the subject matter of the claims. The disclosures of allpatents, patent applications and publications cited herein are herebyincorporated herein by reference, to the extent that they are consistentwith the present disclosure set forth herein.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

A pilot study was conducted to provide proof of principle that fluidshear stress can be used to condition stem cells, alter gene expression,and enhance functional activities. To exemplify the conditioningactivity of shear stress of the type provided by the present system, buton a much smaller analytical scale, force (shear stress) was appliedusing custom fabricated slides or IBIDI® small-scale microfluidicschannel slides obtained from IBIDI, LLC. (Verona, Wis., USA). Humansamples were harvested from bone marrow (BM), amniotic fluid (AF), oradipose (AD) tissue, processed for isolation and expansion of hMSC, andstored frozen. Frozen hMSCs were thawed and seeded into T225 cellculture flasks with 50 ml Minimum Essential Medium (MEM-α) (20% FBS, 5%Penicillin/Streptomycin, 5% Glutamine). The media was replaced every 3-4days. hMSCs were maintained in culture until they were almost 100%confluent. The cells had a fibroblastic phenotype. Prior to seeding thecells onto the device (IBIDI® microfluidic channel slide or a customfabricated slide) to provide fluid laminar shear stress similar to, buton a far more limed scale than that which is provided by the instantsystem, the culture surfaces were pre-coated with 100 ug/ml fibronectinin PBS for 30-45 min at 37° C. and washed 2× with PBS before seedingcells and allowed to sit in the incubator for 30-45 minutes while cellswere prepared for seeding. Cultured hMSC were prepared by removing themedia from the T225 flask using a vacuum and glass Pasteur pipette, thecells were washed 1× with room temperature PBS, which was removed byaspiration. 3 ml of a 0.25% trypsin solution was added and the flask wasincubated at 37° C. for 5 min. Following this incubation, the flask wasremoved from the incubator and tapped vigorously to dislodge the cells.The flask was examined under a dissecting microscope to ensure that allof the cells had detached and were free-floating. At this point 9 ml ofMEM-α was added to the flask and the total volume (12 ml) was removedand placed in a 15 ml conical tube. The tube was placed in a centrifugeand spun at 300 RCF for 5 minutes at room temperature. The supernatantwas aspirated, leaving a small amount of media above the cell pellet, 3ml MEM-α was added and the cell pellet was resuspended in this media.The number of live cells present was determined using trypan blue dyeexclusion. Live cell counts were determined on a hemacytometer and thecells were resuspended to obtain the desired concentration for eachassay (see Table 1). IBIDI channels were utilized to provide fluidlaminar shear stress similar to, but less well controlled than, the typeprovided by the instant system. Cells were allowed to sit for 30-45minutes before filling wells with 60 ul media per well (if too much timepasses, the media will begin to evaporate from the channels). Cells wereallowed to incubate for 12-18 hours. Tubing was then attached to theIBIDI® slide in the safety cabinet with a clean three way stopcock (allpreviously autoclaved or EtO sterilized, the three stop tubing requiredfor use with a peristaltic pump may be EtO or UV sterilized) and thentransferred to incubator. IBIDI channel experiments recirculated a totalvolume of 6 ml and large fabricated slide experiments recirculated 50 mltotal volume. A peristaltic or Harvard syringe pump was programmed topush media across the culture surface at 15 dyne/cm2. Fluid shear stresswas applied for 3, 6 or 8 hours.

TABLE 1 Cell Concentration Culture Total Volume in Time of Shear Assay(cells/ml) Platform channel Exposure qRT PCR 3 × 10{circumflex over( )}6 IBIDI 30 ul 3, 6, 8 hours Western Blot 3 × 10{circumflex over( )}6 IBIDI 30 ul 8 hours NF-kB Binding 2 × 10{circumflex over ( )}6Custom 10 ml 8 hours Assay In Vivo Rat 2 × 10{circumflex over ( )}6Custom 10 ml 8 hours Experiments ELISA assay 2 × 10{circumflex over( )}5 IBIDI 30 ul 3, 8 hours IF Staining 2 × 10{circumflex over ( )}5IBIDI 30 ul 3 hoursImmunomodulatory Changes Due to Fluid Laminar Shear Stress

Naive MSCs do not express key mediators of immunosuppression, such asthe multifunctional anti-inflammatory proteins such as TNF-α stimulatedprotein 6 (TSG-6), prostaglandin E2 (PGE2), and interleukin (IL)-1receptor antagonist (IL1RN). MSCs derived from three human tissuesources, bone marrow, adipose, and amniotic fluid, were all found to beresponsive to shear stress to varying extents. For example, a shearstress applied at a force of 15 dyne/cm² activated immunomodulatorysignaling in MSCs collected from multiple human tissues. Evaluation ofbone marrow-derived MSCs, laminar shear stress stimulated profoundup-regulation, from 6- to 120-fold increases, in transcription of MSCgenes encoding TSG-6, COX-2, IL1RN, HMOX-1, LIF, and KLF2. See forexample (FIG. 20).

Similarly it was determined utilizing commercially available ELISAs thatthe media from human MSCs cultures that had been subject to fluid shearstress also contained immunomodulatory proteins, such as prostaglandinE2. In addition, Western blotting confirmed elevated protein levels(translation) of COX2, TSG6, and IL1RN. Actin protein expression levels,which are constant, were used as a control for baseline proteinexpression. In this study, it was determined that after exposure ofhuman MSC (derived from hBM, human bone marrow MSC; hAF MSC, amnioticfluid MSC; hAD MSC, adipose-derived MSC) to 8 hours of fluid shearstress, there was a significant increase in the expression of COX2protein as compared with media obtained from MSC that were not subjectto fluid shear stress. It was also determined that this induction couldbe abrogated by the addition of 10 uM of the NF-kappaB antagonistBAY11-7085 (FIG. 21). Furthermore by utilizing commercially availableELISAs, it was determined that the media from human MSCs cultures thathad been subject to fluid shear stress for as little as 3 hours hadimmunosuppressive activity, as evidenced by the 10-50% reduction inTNF-α when treated hMSCs were co-cultured with activated immune cellsfrom the spleen (FIG. 22). It was also determined using cytokinesuppression assays that application of COX or NF-kB inhibitors abrogatedthe ability of shear treated MSC to suppress TNF-α production; whereas,addition of a stabilized synthetic form of PGE2 (dmPGE2) reduced TNF-αto levels produced in the presence of sheared MSCs (FIG. 23). Additionalevidence further suggests that sheared MSCs may be more responsive toother priming agents than MSC that have not been subject to a fluidshear stress, as a greater induction of COX2 and HMOX1 occurred with theaddition of IFN-γ (FIG. 24). Thus, indicating that human MSC subject tofluid shear stress may act synergistically with cytokines when presentedin, for example, combination therapies.

It was further determined that naive MSCs exposed to shear stress, withno preconditioning by inflammatory cytokines, were capable of blockingTNF-α secretion by lipopolysaccharide (LPS)-activated mouse splenocytes(ranging from complete inhibition to 2-fold reduction below MSC culturedunder static conditions, depending upon MSC donor and sourcevariability).

Neuroprotective Abilities:

Studies were performed to establish that cells such as MSC exposed tocontrolled shear stress can provide neuroprotection following, forexample, traumatic brain injury (TBI). To do this a rat model wasutilized to evaluate functional outcomes. Controlled cortical impact(CCI) in the rat presents morphologic and cerebrovascular injuryresponses that resemble human head trauma. Thus, characterization of thecellular and molecular alterations that potentiate neurological damageand inflammation provides a powerful tool to measure potential clinicalefficacy of MSC preconditioning.

Twelve (12) rats were predicted to be necessary per condition to achieve80% power, at an alpha error level of 0.05 (SAS predictive analyticssoftware). Cells to be administered for cellular therapy were bonemarrow-derived MSCs. MSCs were exposed to static conditions or to shearstress for 3 hrs at an intensity of 15 dyne/cm2, a fluid flow rate andduration demonstrated to produce robust induction of COX2, TSG6, IL1RN,and HMOX1 and to suppress cytokine production in activated immune cells.Immediately following application of force by the large capacity lateralflow system, 10×10⁶ cells/kg MSCs were transferred to recipient rats viatail vein injection (approximate dose of 2.5×10⁶ MSCs per rat).

Blood-brain barrier (BBB) permeability was determined using standardmethods for examining leakage across the vasculature (utilizing dextranbeads in suspension as described herein). Injury at the right parietalassociation cortex was introduced in male rats (225-250 grams) by a CCIdevice (Leica Impactor 1). In parallel, control rats were treated withCCI alone or simply anesthetized (sham control). Forty eight (48) hoursafter injury, the MSCs were administered. Twenty-four (24) hrs after theinjection of MSCs, fluorescently conjugated Alexa 680-dextran beads (10kDa, 0.5 ml of 1 mg/ml) were delivered via tail vein. Thirty (30)minutes after this dye was injected, animals were euthanized andperfused with 4% paraformaldehyde. Fixed brains were sectioned coronallyat 1 mm thickness. Vascular leakage was measured by fluorescenceintensity of brain sections in a LI-COR Odyssey CLx infrared laserscanner using 700 and 800 nm channels (800 nm signal was used forbackground subtraction). Histological analysis of the frequencies ofcertain immune and neural cell types that are known to be rapidlyaltered in response to neuroinflammation and are important indicators ofprognosis. In future studies, in an independent cohort of rats,

brain sections of between 8 to 50 um will be analyzed for inflammatoryphenotypes in the CNS by immunohistochemistry using antibodies to detectmicroglia (Iba1, ED1 or CD63), infiltrating neutrophils (RP-3),astroglia (GFAP), and neurons (NeuN), as well as an indicator of celldeath (cleaved caspase 3). Staining of brain sections will be done usinga standard free floating staining protocol or slide-mountedcryosections.

Cognitive recovery of treated and control rats can be assessed by aclassic hippocampus-dependent spatial learning task, the Morris watermaze, in which rats locate an underwater platform on the basis ofextra-maze cues. For these studies, two (2) weeks following injury,learning is measured by speed, time spent in each quadrant, and distanceof the path taken to find the platform. The same individuals will betested at 4 weeks post injury for memory function by the same measuresin the maze. The anticipated results are that delivery of sheared MSCswill reduce BBB permeability and inflammatory cell phenotypes in thebrain relative to naïve static-cultured MSC and will also result inimproved cognitive recovery.

Example 2 Injury Alters the Frequency of MSCs in the Bone Marrow

Chronic inflammation in traumatic brain injury is perpetuated bymonocytes and lymphocytes of the innate and adaptive immune systems.MSCs are reported to home from the bone marrow to sites of injury andinflammation, yet detailed analysis of MSC trafficking from the marrowin this context is lacking. To monitor changes in MSC frequency causedby injury, a rat model of traumatic brain injury was established whichpresents morphologic and cerebrovascular injury responses that resemblehuman head trauma. Briefly, controlled cortical impact (CCI) wasdelivered to the exposed right parietal association cortex adjacent tothe midline suture. In parallel, sham controls were anesthesized andincisions were made without injury. The frequency of CD105+ MSCs wereexamined within the bone marrow and it was found that the absolutenumber of CD105+ MSCs was significantly decreased 24 hours after injury(FIGS. 25A and 25B). These data suggest that MSCs respond to injury byegress from the bone marrow, much as might be observed by hematopoieticstem or progenitor cells, thus increasing the likelihood that MSCs areexposed directly to hemodynamic forces present within the blood streamand on the vascular wall. Importantly, intravenous administration ofhuman MSCs preconditioned by 3 hours of 15 dyne/cm² WSS significantlyelevated CD105+ MSC frequency in the bone marrow of injured rats whenadministered 24 hours after CCI (FIGS. 25C and 25D). At 72 hourspost-CCI, CD105+ cell frequency was significantly higher when eitherstatic cultured or WSS-exposed MSCs were administered, and theprotective effect on the bone marrow was significantly greater when MSCswere preconditioned by WSS.

Materials and Methods

Cell Culture—Bone marrow MSCs were derived from whole bone marrow fromindependent human donors (AllCells). Briefly, mononuclear cells wereenriched in the buffy layer of whole bone marrow by phase separation inFicoll-Paque. Cells were either cryopreserved or resuspended forimmediate expansion in complete culture medium consisting of MEM-α(Thermo Scientific), 20% fetal bovine serum (Atlanta Biologicals), 100units/ml penicillin (Gibco), 100 pg/ml streptomycin (Gibco), and 2 mML-glutamine (Gibco). Nonadherent cells were removed after 2 days.Adherent colonies were expanded further and frozen as Passage 1. ThawedMSCs were plated at 1×10⁵ cells/ml, and medium was changed every threedays. At 80% confluence, cells were passaged into IBIDI channels(μ-Slide VI 0.4) at a density of 3×10⁶ cells/ml for qRT PCR,immunoblotting, and rat CCI experiments and at 5×10⁵ cells/ml for ELISAand immunofluorescence experiments. Following attachment to the culturesurface, syringe pumps (PhD ULTRA programmable, Harvard Apparatus) orperistaltic pumps (REGLO analog MS4/12, Ismatec) were used to producelaminar shear stress of 15 dyne/cm².

Controlled cortical impact (CCI)—A CCI device (Leica IMPACT ONE™) wasused to deliver a single impact of 3.1 mm compression at 6 m/sec at theright parietal association cortex (adjacent to midline suture betweenbregma & lambda) using a 6 mm impactor tip on exposed brain in male rats(225-250 grams). In parallel, control rats were treated with CCI aloneor simply anesthesized (sham control). In cell therapy experiments, lowpassage (P2-5) MSCs were cultured under static conditions or shearstress of 15 dyne/cm² for 3 hrs. Recipient rats received MSCs (10×10⁶cells/kg) via tail vein within 2 hrs of shear exposure. At time ofsacrifice, rats were perfused with 4% paraformaldehyde and tissues werefurther fixed after collection. All experiments were conducted incompliance with guidelines from the University of Texas Health ScienceCenter Institutional Animal Care and Use Committee.

Histological processing of rat tissues—Rat tibias were harvested and themuscle surrounding the bone was carefully removed. The bones werefurther fixed in 4% paraformaldehyde and decalcified using 10% EDTA.Once decalcified, the bones were transferred to the Histology Core at UTMedical School for grossing, paraffin embedding, and sectioning.

Immunostaining of bone barrow—Paraffin embedded sections were baked at60° C. for 20 mins and serially rehydrated in xylene and varying gradesof ethanol. Heat-induced epitope retrieval was performed using DAKOTarget Antigen Retrieval Solution (pH 6.1). Endogenous peroxidase wasblocked with 0.3% H₂O₂. Slides were blocked with 2.5% BSA for 1 hr andincubated overnight with anti-CD105 antibody (1:200, SN6, Ab11414) in2.5% BSA. Immunoperoxidase detection was performed using the Vector ABCkit and DAKO DAB kit (VECTASTAIN® Elite ABC kit; PK-6102, Dako DAB kit;K3468) according to manufacturer instructions and counterstained withNuclear Fast Red or CAT Hemotoxylin (Vector Labs; H-3403, BiocareMedical; 012215). To ensure optimal cellular contrast a bluing reagentwas used with Hemotoxylin (Statlab, SL203). The slides were air driedand coverslipped with Biocare Ecomount (EM897L).

Image Acquisition and Analysis—Photomicrographs for immunohistochemistrywere acquired with an Olympus BX51P polarizing microscope (DP71 ColorCamera) and DP Controller software (Olympus, Verson 3.3.1.292). Imageswere quantitatively analysed using Image J (NIH). CD105+ MSCs werecounted by an investigator blinded to the treatment group and sampleidentification.A random number generator was used to select 8 randomimage sets which were subsequently subjected to statistical analysis.

Statistical analyses—All data were analyzed with SIGMAPLOT® 12.5software for statistical significance and are reported as mean±SEM.One-way ANOVA and the Holm-Sidak method for multiple comparisons wereused to evaluate differences in histological measurements. Significancelevels of P<0.001 are denoted in graphs by a triple asterisk ***.Representative results from at least three independent biologicalreplicates are shown unless stated otherwise. (See FIGS. 25B and 25D)

* * *

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated herein byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines a termin a manner that contradicts the definition of that term in thisapplication, this application controls.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   International Publication No. WO98/30679-   Wagner et al., Optimizing mesenchymal stem cell-based therapeutics.    Curr Opin Biotechnol 20(5): 531-536, 2009.

We claim:
 1. A cell preparation system comprising: a base; and a plurality of modular cell preparation apparatus coupled to the base, wherein each modular cell preparation apparatus comprises: an inlet fluid feed plate; a base plate; a first conduit; a second conduit; a plurality of intermediate plates positioned between the inlet fluid feed plate and the base plate; a reservoir; and a pump, wherein: the inlet fluid feed plate comprises a first fluid inlet, a first fluid outlet, a second fluid inlet and a second fluid outlet; the reservoir is coupled to the first fluid inlet via the first conduit; and the reservoir is coupled to the second fluid outlet via the second conduit.
 2. The cell preparation system of claim 1 wherein the plurality of modular cell preparation apparatus comprises at least three modular cell preparation apparatus.
 3. The cell preparation system of claim 1 wherein during use the plurality of modular cell preparation apparatus are configured to operate in parallel to prepare cells.
 4. The cell preparation system of claim 1 wherein the plurality of intermediate plates comprises at least three intermediate plates.
 5. The cell preparation system of claim 1 wherein each of the intermediate plates in the plurality of intermediate plates comprises: a first end, a second end, a first side, and a second side; a distribution channel proximal to the first end; and a gathering channel proximal to the second end, wherein: the distribution channel extends between the first side and the second side of the intermediate plate; and the gathering channel extends between the first side and the second side of the intermediate plate.
 6. The cell preparation system of claim 5 wherein: the distribution channel of each intermediate plate has a first length; the gathering channel of each intermediate plate has a second length; each of the intermediate plates has a width between first side and a second side; the first length of the distribution channel extends across a majority of the width of the intermediate plate; and the second length of the gathering channel extends across a majority of the width of the intermediate plate.
 7. The cell preparation system of claim 5 wherein: the distribution channel of each intermediate plate has a first length; the gathering channel of each intermediate plate has a second length; each of the intermediate plates has a width between first side and a second side; the first length of the distribution channel extends across at least 70 percent of the width of the intermediate plate; and the second length of the gathering channel extends across at least 70 percent of the width of the intermediate plate.
 8. The cell preparation system of claim 1 wherein the pump of each modular cell preparation apparatus is coupled to the first fluid outlet and the second fluid inlet of the inlet fluid feed plate.
 9. The cell preparation system of claim 1 wherein the pump comprises: a rolling element; and a flexible conduit, wherein the flexible conduit is coupled to the first fluid outlet and the second fluid inlet of the inlet fluid feed plate.
 10. The cell preparation system of claim 9 further comprising an electric motor coupled to the rolling element.
 11. The cell preparation system of claim 10 wherein a velocity of the fluid through the second fluid inlet is controlled by a rotational speed of the rolling element of the pump.
 12. The cell preparation system of claim 9 wherein during use the pump is configured to: draw a fluid from the reservoir through first fluid inlet and the first fluid outlet; direct the fluid through the second fluid inlet, across the plurality of intermediate plates, out the second outlet, and back to the reservoir. 